Gold nanoparticles enhance cisplatin delivery and potentiate chemotherapy by decompressing colorectal cancer vessels

Abstract

Background: Tumor vessels were persistently compressed by solid stress from tumor interstitial matrix, resulting in limited vessel perfusion and oxygen concentrations. Collagen within matrix participated in transmitting the solid stress to tumor vessels and limiting drug delivery.

Purpose: The objective of this study was to identify whether gold nanoparticles (AuNPs) were able to decompress colorectal cancer vessels and enhance vessel perfusion as well as drug delivery in colorectal cancer.

Methods: Colorectal cancer xenograft mice were treated with AuNPs or normal saline for 14 days. The cancer stromal collagen I level, cancer vessel perfusion, hypoxia of tumor were tested by histological examination. We also test the solid stress in the two groups. Furtherly, the effect and the drug delivery of combined using AuNPs and cisplatin were tested. The effect and the underlying mechanism of AuNPs on SW620 cells were tested by CCK8, flow cytometry, Western-blot and atomic force microscope.

Results: AuNPs were able to decrease the density of colorectal cancer associated fibroblasts (CAFs), to reduce the production of tumor stromal collagen I, and to diminish the expression of profibrotic signals, including CTGF, TGF-β1 as well as VEGF in vivo and vitro via Akt signaling pathway. Consequently, AuNPs could alleviate solid stress in tumors, subsequently leading to enhanced vessel perfusion. Therefore, cisplatin as well as oxygen delivery to tumors were improved by AuNPs, which reduced hypoxia while sensitizing therapy of cisplatin in colorectal cancer model.

Conclusion: AuNPs were effective agents in enhancing cisplatin delivery and potentiating inhibiting tumor growth by decompressing colorectal cancer vessels.

Keywords: colorectal cancer; decompression; gold nanoparticles; hypoxia; solid stress; vessel.

Figure 1

Characterization of AuNPs. Notes: (A, B) FETEM images reveal discrete particles of AuNPs. Scale bar: 200 nm and 100 nm. (C) UV-visible spectra of AuNPs. The UV-visible spectra of 15 nm AuNPs were characterized by surface plasma resonance absorption peaks at approximately 517 nm. Abbreviations: AuNPs, gold nanoparticles; FETEM, field-emission transmission electron microscopy; UV, ultraviolet.

Figure 2

AuNPs decreased stromal collagen I level in SW620 tumor. (A) Immunofluorescence images of the effect of AuNPs on stromal collagen I (green) level in SW620 tumor. Scale bar, 100 µm. (B) Quantification of tumor collagen I level after the administration of AuNPs. AuNPs reduced the proportion of collagen I-positive region in SW620 tumor (n=6, ^***P<0.001, Student’s t-test). Note: Error bars indicate SEM. Abbreviations: AuNPs, gold nanoparticles; Ctrl, control; SEM, standard error of the mean.

Figure 3

AuNPs decreased profibrotic cytokine levels in plasma and SW620 tumor. Notes: (A) Plasma level of TGF-β1 was decreased after treatment with AuNPs (n=6, ^***P<0.001). (B) Plasma level of CTGF was decreased (n=6, ^***P<0.001). (C) The plasma level of VEGF was reduced after the administration of AuNPs (n=6, ^***P<0.001). (D) Histologic images of the effect of AuNPs on TGF-β1, CTGF, and VEGF expression in SW620 tumor (n=6). Scale bar, 100 µm. AuNPs decreased the expression of TGF-β1, CTGF, and VEGF (^***all P<0.001) in SW620 tumor. Student’s t-test was used for analysis, and error bars indicate SEM. Abbreviations: AuNPs, gold nanoparticles; CTGF, connective tissue growth factor; Ctrl, control; SEM, standard error of the mean; VEGF, vascular endothelial growth factor.

Figure 4

AuNPs reduced α-SMA-positive CAF density in SW620 tumor. Notes: (A) Histologic imaging illustrating the effect of AuNPs on the level of α-SMA-positive CAFs (brown) in tumor tissues. Scale bar, 100 µm. (B) Immunohistochemical analysis showed that the administration of AuNPs diminished the α-SMA-positive CAF density in SW620 tumor (n=6, ^**P=0.0032, Student’s t-test). Error bars indicate SEM. Abbreviations: AuNPs, gold nanoparticles; α-SMA; alpha-smooth muscle actin; CAFs, cancer-associated fibroblasts; Ctrl, control; SEM, standard error of the mean.

Figure 5

AuNPs reduced solid stress in SW620 tumor. Notes: An ex vivo technology was employed to evaluate tumor solid stress by detecting the degree of tumor tissue relaxation (tumor opening relative to tumor diameter) after a stress-releasing incision, with larger openings indicating higher stress. Through its antimatrix effects, AuNPs decreased solid stress in SW620 tumor (n=6, ^***P=0.0002, Student’s t-test). Error bars indicate SEM. Abbreviations: AuNPs, gold nanoparticles; Ctrl, control; SEM, standard error of the mean.

Figure 6

AuNPs increased vascular perfusion as well as drug delivery and reduced tumor hypoxia in SW620 tumor. Notes: (A) Representative pictures of perfused tumor vessels (green) under immunofluorescence microscopy. The density of perfused vessels was elevated by the administration of AuNPs in SW620 colorectal cancer models (n=6, ^**P=0.0026). Scale bar, 100 µm. (B) Pimonidazole injection and staining demonstrated that the hypoxic fraction was decreased by the administration of AuNPs in SW620 tumor (n=6, ^***P<0.001) due to the increased oxygen supply. Scale bar, 100 µm. (C) Necrotic areas in the tumoral regions measured by H&E staining following treatment with AuNPs. AuNPs reduce necrotic areas in SW620 tumors (n=6, ^***P<0.001). Scale bar: 100 µm. Cisplatin delivery to the tumor (D), liver (E), and kidney (F) after treatment with AuNPs. AuNPs promoted the cisplatin accumulation in SW620 colorectal cancer models (n=3, ^**P=0.0023), which, however, did not affect cisplatin accumulation in the liver (n=3, P=0.2365) or the kidney (n=3, P=0.3314). Student’s t-test was utilized for analysis, and error bars indicate SEM. Abbreviations: AuNPs, gold nanoparticles; Ctrl, control; SEM, standard error of the mean.

Figure 7

AuNPs potentiated cisplatin efficacy. Notes: (A) Quantification of tumor volume at different times in mice models treated with AuNPs, cisplatin, combination of AuNPs and cisplatin, or saline. Both cisplatin and AuNPs monotherapy significantly inhibited tumor growth compared with the control treatment, and tumor growth was more greatly inhibited by a combined treatment of cisplatin and AuNPs (n=6). (B) Mouse body weight of the four groups was monitored before and after treatment in different times. (C) Representative images of liver and kidney were removed from the four groups. (D–E) The effect of AuNPs, cisplatin, and combination treatment on the levels of serum IL-6 and TNF-α of mice. The serum IL-6 and TNF-α levels in the cisplatin and combination group were significantly increased comparing with the control and AuNPs groups at day 1, day 5, and day 10 (^*compared with the control group, P<0.05; ^#compared with the AuNPs group, P<0.05). One-way ANOVA was utilized for analysis, and error bars indicate SEM (^*P<0.05, ^***P<0.001). Abbreviations: AuNPs, gold nanoparticles; Ctrl, control; SEM, standard error of the mean.

Figure 8

AuNPs reduced TGF-β1, CTGF, and VEGF production via Akt-dependent pathway. Notes: (A) Cell proliferation rate after AuNP treatment with different concentrations was detected by CCK-8 assay (n=6). (B, C) The effect of different treatments on the apoptosis of SW620 cells, no significant difference of the apoptosis rate was found after AuNP treatment with different concentrations (n=3). (D) The effects of AuNPs with different concentrations on TGF-β1, CTGF, and VEGF expression of SW620 cells were determined using Western blot analysis. (E) The effects of AuNPs on the expression of Akt and phosphorylated Akt were detected by Western blot analysis. (F–H) The effects of AuNPs with different concentrations on TGF-β 1, CTGF, and VEGF secretion of SW620 cells were determined using ELISA analysis (n=3; AuNPs1: 10 ng/mL; AuNPs2: 25 ng/mL; AuNPs3: 50 ng/mL; AuNPs4: 100 ng/mL). One-way ANOVA was utilized for analysis, and error bars indicate SEM (^***P<0.001 vs Ctrl group). Abbreviations: AuNPs, gold nanoparticles; CTGF, connective tissue growth factor; Ctrl, control; SEM, standard error of the mean; VEGF, vascular endothelial growth factor.

Figure 9

AFM images demonstrate the uptake of AuNPs by SW620 cells. Notes: (A) Representative morphology images of SW620 cells of the control group and AuNPs group. (B) Representative cellular surface ultrastructure images of SW620 cells (AuNPs1: 10 ng/mL; AuNPs2: 25 ng/mL). Abbreviations: AFM, atomic force microscope; AuNPs, gold nanoparticles; Ctrl, control.